The objective of this research is to elucidate the control mechanisms involved in the regulation of de novo pyrimidine biosynthesis in mammalian cells. The first three enzymes of the pathway are associated with a single polypeptide chain. This large multifunctional protein, CAD, also exhibits allosteric transitions. The protein isolated from an over-producing mutant SV40-transformed cell line is being cleaved by controlled proteolysis to determine the number, function and arrangement of autonomously folded structural domains. Thus far, a 40 Kd and a 44 Kd species corresponding to the aspartate transcarbamylase and dihydroorotase domains, respectively, have been isolated. A 150 Kd species has also been identified that carries a fully functional carbamyl phosphate synthetase domain and all of the regulatory elements of the complex. The proteolytic fragments will be characterized by analytical peptide mapping, end group analysis, amino acid composition and partial sequencing by HPLC. Both poly-\and monclonal antibodies will be used to investigate the domain composition of the larger fragments, for isolation by immunoaffinity chromatography and as structural probes. The stucture and interconversion of the oligomeric forms of the complex and isolated domains will be investigated. Functional studies of the parent molecule, the isolated domains and reconstituted species will focus on aggregate properties, such as channeling, reduction in transient time and coordinate effects and their interrelationship to the allosteric control mechanisms.